Fibroblast growth factor receptor 3 alterations and response to immune checkpoint inhibition in metastatic urothelial cancer: a real world experience.

BACKGROUND: FGFR3-altered urothelial cancer (UC) correlates with a non-T cell-inflamed phenotype and has therefore been postulated to be less responsive to immune checkpoint blockade (ICB). Preclinical work suggests FGFR3 signalling may suppress pathways such as interferon signalling that alter immune microenvironment composition. However, correlative studies examining clinical trials have been conflicting as to whether FGFR altered tumours have equivalent response and survival to ICB in patients with metastatic UC. These findings have yet to be validated in real world data, therefore we evaluated clinical outcomes of patients with FGFR3-altered metastatic UC treated with ICB and investigate the underlying immunogenomic mechanisms of response and resistance. METHODS: 103 patients with metastatic UC treated with ICB at a single academic medical center from 2014 to 2018 were identified. Clinical annotation for demographics and cancer outcomes, as well as somatic DNA and RNA sequencing, were performed. Objective response rate to ICB, progression-free survival, and overall survival was compared between patients with FGFR3-alterations and those without. RNA expression, including molecular subtyping and T cell receptor clonality, was also compared between FGFR3-altered and non-altered patients. RESULTS: Our findings from this dataset confirm that FGFR3-altered (n = 17) and wild type (n = 86) bladder cancers are equally responsive to ICB (12 vs 19%, p = 0.73). Moreover, we demonstrate that despite being less inflamed, FGFR3-altered tumours have equivalent T cell receptor (TCR) diversity and that the balance of a CD8 T cell gene expression signature to immune suppressive features is an important determinant of ICB response. CONCLUSIONS: Our work in a real world dataset validates prior observations from clinical trials but also extends this prior work to demonstrate that FGFR3-altered and wild type tumours have equivalent TCR diversity and that the balance of effector T cell to immune suppression signals are an important determinant of ICB response.

Enhancing Next-Generation Sequencing-Guided Cancer Care Through Cognitive Computing.

BACKGROUND: Using next-generation sequencing (NGS) to guide cancer therapy has created challenges in analyzing and reporting large volumes of genomic data to patients and caregivers. Specifically, providing current, accurate information on newly approved therapies and open clinical trials requires considerable manual curation performed mainly by human "molecular tumor boards" (MTBs). The purpose of this study was to determine the utility of cognitive computing as performed by Watson for Genomics (WfG) compared with a human MTB. MATERIALS AND METHODS: One thousand eighteen patient cases that previously underwent targeted exon sequencing at the University of North Carolina (UNC) and subsequent analysis by the UNCseq informatics pipeline and the UNC MTB between November 7, 2011, and May 12, 2015, were analyzed with WfG, a cognitive computing technology for genomic analysis. RESULTS: Using a WfG-curated actionable gene list, we identified additional genomic events of potential significance (not discovered by traditional MTB curation) in 323 (32%) patients. The majority of these additional genomic events were considered actionable based upon their ability to qualify patients for biomarker-selected clinical trials. Indeed, the opening of a relevant clinical trial within 1 month prior to WfG analysis provided the rationale for identification of a new actionable event in nearly a quarter of the 323 patients. This automated analysis took <3 minutes per case. CONCLUSION: These results demonstrate that the interpretation and actionability of somatic NGS results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing could potentially improve patient care by providing a rapid, comprehensive approach for data analysis and consideration of up-to-date availability of clinical trials. IMPLICATIONS FOR PRACTICE: The results of this study demonstrate that the interpretation and actionability of somatic next-generation sequencing results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing can significantly improve patient care by providing a fast, cost-effective, and comprehensive approach for data analysis in the delivery of precision medicine. Patients and physicians who are considering enrollment in clinical trials may benefit from the support of such tools applied to genomic data.

Tumor Cell Extrinsic Synaptogyrin 3 Expression as a Diagnostic and Prognostic Biomarker in Head and Neck Cancer.

Over 70% of oropharyngeal head and neck squamous cell carcinoma (HNSC) cases in the United States are positive for human papillomavirus (HPV) yet biomarkers for stratifying oropharyngeal head and neck squamous cell carcinoma (HNSC) patient risk are limited. We used immunogenomics to identify differentially expressed genes in immune cells of HPV(+) and HPV(-) squamous carcinomas. Candidate genes were tested in clinical specimens using both quantitative RT-PCR and IHC and validated by IHC using the Carolina Head and Neck Cancer Study (CHANCE) tissue microarray of HNSC cases. We performed multiplex immunofluorescent staining to confirm expression within the immune cells of HPV(+) tumors, receiver operating characteristic (ROC) curve analyses, and assessed survival outcomes. The neuronal gene Synaptogyrin-3 (SYNGR3) is robustly expressed in immune cells of HPV(+) squamous cancers. Multiplex immunostaining and single cell RNA-seq analyses confirmed SYNGR3 expression in T cells, but also unexpectedly in B cells of HPV(+) tumors. ROC curve analyses revealed that combining SYNGR3 and p16 provides more sensitivity and specificity for HPV detection compared to p16 IHC alone. SYNGR3-high HNSC patients have significantly better prognosis with five-year OS and DSS rates of 60% and 71%, respectively. Moreover, combining p16 localization and SYNGR3 expression can further risk stratify HPV(+) patients such that high cytoplasmic, low nuclear p16 do significantly worse (Hazard Ratio, 8.6; P = 0.032) compared to patients with high cytoplasmic, high nuclear p16. SYNGR3 expression in T and B cells is associated with HPV status and enhanced survival outcomes of HNSC patients.

UNMASC: tumor-only variant calling with unmatched normal controls.

Despite years of progress, mutation detection in cancer samples continues to require significant manual review as a final step. Expert review is particularly challenging in cases where tumors are sequenced without matched normal control DNA. Attempts have been made to call somatic point mutations without a matched normal sample by removing well-known germline variants, utilizing unmatched normal controls, and constructing decision rules to classify sequencing errors and private germline variants. With budgetary constraints related to computational and sequencing costs, finding the appropriate number of controls is a crucial step to identifying somatic variants. Our approach utilizes public databases for canonical somatic variants as well as germline variants and leverages information gathered about nearby positions in the normal controls. Drawing from our cohort of targeted capture panel sequencing of tumor and normal samples with varying tumortypes and demographics, these served as a benchmark for our tumor-only variant calling pipeline to observe the relationship between our ability to correctly classify variants against a number of unmatched normals. With our benchmarked samples, approximately ten normal controls were needed to maintain 94% sensitivity, 99% specificity and 76% positive predictive value, far outperforming comparable methods. Our approach, called UNMASC, also serves as a supplement to traditional tumor with matched normal variant calling workflows and can potentially extend to other concerns arising from analyzing next generation sequencing data.

Correction: Molecular Subtypes in Head and Neck Cancer Exhibit Distinct Patterns of Chromosomal Gain and Loss of Canonical Cancer Genes.

[This corrects the article DOI: 10.1371/journal.pone.0056823.].

The Prognostic Significance of Low-Frequency Somatic Mutations in Metastatic Cutaneous Melanoma.

Background: Little is known about the prognostic significance of somatically mutated genes in metastatic melanoma (MM). We have employed a combined clinical and bioinformatics approach on tumor samples from cutaneous melanoma (SKCM) as part of The Cancer Genome Atlas project (TCGA) to identify mutated genes with potential clinical relevance. Methods: After limiting our DNA sequencing analysis to MM samples (n = 356) and to the CANCER CENSUS gene list, we filtered out mutations with low functional significance (snpEFF). We performed Cox analysis on 53 genes that were mutated in ≥3% of samples, and had ≥50% difference in incidence of mutations in deceased subjects versus alive subjects. Results: Four genes were potentially prognostic [RAC1, FGFR1, CARD11, CIITA; false discovery rate (FDR) < 0.2]. We identified 18 additional genes (e.g., SPEN, PDGFRB, GNAS, MAP2K1, EGFR, TSC2) that were less likely to have prognostic value (FDR < 0.4). Most somatic mutations in these 22 genes were infrequent (< 10%), associated with high somatic mutation burden, and were evenly distributed across all exons, except for RAC1 and MAP2K1. Mutations in only 9 of these 22 genes were also identified by RNA sequencing in >75% of the samples that exhibited corresponding DNA mutations. The low frequency, UV signature type and RNA expression of the 22 genes in MM samples were confirmed in a separate multi-institution validation cohort (n = 413). An underpowered analysis within a subset of this validation cohort with available patient follow-up (n = 224) showed that somatic mutations in SPEN and RAC1 reached borderline prognostic significance [log-rank favorable (p = 0.09) and adverse (p = 0.07), respectively]. Somatic mutations in SPEN, and to a lesser extent RAC1, were not associated with definite gene copy number or RNA expression alterations. High (>2+) nuclear plus cytoplasmic expression intensity for SPEN was associated with longer melanoma-specific overall survival (OS) compared to lower (≤ 2+) nuclear intensity (p = 0.048). We conclude that expressed somatic mutations in infrequently mutated genes beyond the well-characterized ones (e.g., BRAF, RAS, CDKN2A, PTEN, TP53), such as RAC1 and SPEN, may have prognostic significance in MM.

Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC.

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07-0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11-1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.

Molecular subtypes in head and neck cancer exhibit distinct patterns of chromosomal gain and loss of canonical cancer genes.

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.